The following publications are referred to by corresponding number in this application:
1. Fry, J. M., Lisak, R. P., Manning, M. C., and Silberberg, D. H., J. Immunol. Methods 11:185-193 (1976). PA1 2. U.S. Pat. No. 4,342,739, issued Aug. 3, 1982 to Kakimi, F. et al. PA1 3. Szoka, F. Jr., and Papahadjopoulos, D., Ann. Rev. Biophys. Bioeng. 9:467-508 (1980). PA1 4. Szoka, F., Jr. and Papahadjopoulos, D., Proc. Nat. Acad. Sci. USA 75:4194-4198 (1978). PA1 5. Reeves, J. P. and Dowben, R. M., J. Cell Physiol. 73:49-57 (1969). PA1 6. Hub, H. H., Zimmerman, V., and Ringsdorf, H., FEBS Letters 140 No. 2:254-256 (1982). PA1 7. Lenk, R. P., et al., Eur. J. Biochem. 121:475 (1982). PA1 8. Heath, T. D., Macher, B. A. and Papahadjopoulos, D., Biochimica et Biophysica Acta 640:66-81 (1981). PA1 9. Marin, F. J., Hubbell, W. L., and Papahadjopoulos, D. Biochemistry 20:4229-4238 (1981). PA1 10. Martin, F. J. and Papahadjopoulos, D., J. Biol. Chem. 257:286-288 (1982). PA1 11. Smith, B. A. and McConnell, H. M., Proc. Nat. Acad. Sci. USA 75:2759-2763 (1978).
The present invention relates to a large-liposome agglutination assay reagent, and to a method using such a reagent.
A variety of methods for determining the presence or concentration of biochemical analytes is available. The analyte to be assayed typically is one which plays an important role in biochemical processes, or is diagnostic of a particular disease state.
Several analyte-assay techniques are based on specific, high-affinity binding between the analyte to be assayed and a ligand in an assay reagent. The ligand and analyte are opposite members of a high-affinity, ligand/anti-ligand binding pair, which may include antigen-antibody, immunoglobulin-protein A, carbohydrate-lectin, transport protein-receptor protein, biotin-avidin, hormone-hormone receptor protein, and complementary oligo-and polynucleotide strand pairs.
One general type of assay procedure which is based on specific ligand/analyte binding involves particle agglutination. In a typical agglutination assay, particles coated with ligand molecules are mixed with a multivalent ligand-binding analyte, producing visible particle clumping or aggregation, the extent of which is related to the amount of analyte present. The analyte functions to bridge ligands carried on different ligand-coated particles. Accordingly, the analyte molecules or particles each must have at least two ligand-binding sites which are arranged spatially to promote such bridging. The analyte may be either divalent, meaning it has two such binding sites, or multivalent, meaning that more than two sites are present in the molecule. The term "multivalent" will be used herein to denote analyte molecules or particles containing two or more binding sites capable of bridging ligands carried on separate agglutinatable particles.
A variety of agglutinatable particles may be used in forming an agglutination-assay reagent. For example, the Venereal Disease Research Laboratory (VDRL) test for syphilis uses an agglutination reagent composed of an emulsion of lipid droplets containing the nontreponemal lipid antigen cardiolipin. When the droplet suspension is mixed with the serum of a syphilitic subject, reagin antibody present in the serum as a result of syphilis infection reacts specifically and with high affinity to cardiolipin, producing particle agglutination.
In the VDRL test, a sample of the serum to be tested for the presence of reagin is heat-treated for 30 minutes at about 56.degree. C., and a well-defined quantity of the heat-treated serum is placed as a droplet on the surface of a glass slide or the like. A measured quantity of the lipid suspension is added to the serum aliquot, and the reaction components are mixed by rotating the slide at about 180 rpm on a mechanical rotor. After a 4-minute mixing period, the slide is examined microscopically for evidence of emulsion-particle agglutination.
Because the cardiolipin-containing lipid emulsion is relatively unstable, it must be prepared fresh on the day of use, adding time and expense to the procedure. Preparing and heat-treating the serum sample for use in the test is also time consuming. That the reaction components must be mixed by rotation at a precise mixing speed, and that a microscope must by used for detecting the agglutination reactin also add to the inconvenience and expense of the test.
Lipid bilayer vesicles, or liposomes, have also been proposed in connection with particule-agglutination reagents. U.S. Pat. No. 4,232,001 to Jensen suggests using an estrophylin-liposome agglutination reagent for detection of anti-estrophylin antibody. A serological technique for detection of antibody to galactocerebroside using a galactocerebroside-containing liposome reagent has been described by Fry, et al. in reference 1. In this technique, heat-treated antiserum is incubated with the liposome reagent for about 1 hour, after which the reaction mixture is examined for clumps of agglutinated liposomes. The liposome reagent was prepared by a method which produces multilamellar vesicles of heterogeneous sizes ranging from about 0.05 to 20 microns in diameter.
Liposome agglutination reagents known in the prior art are characterized by relatively slow agglutination times, which may make them impractical for use in clinical testing. Many agglutination assays are conveniently performed as a droplet assay on a glass slide or the like, and evaporation losses from the droplet limit reaction times to about 10 minutes at most. In the above-referenced serological technique described by Fry, et al., for example, a serum sample was incubated with the liposome reagent in a shaking water bath for one hour. The extended reaction time may also accentuate undesired secondary reactions which affect assay results. Such reactions may include analyte instability, non-specific liposome agglutination, and complement-mediated liposome disruption.
Another limitation associated with liposome-agglutination reagents known in the prior art is the difficulty of visualizing clumps of agglutinated liposomes. Although Fry, et al. describe examining for agglutination visually, it has been the experience of the inventors that clumps of agglutinated liposomes produced in accordance with prior art methods can be identified with certainty, particularly in relatively weak agglutination reactions, only by microscopic examination.
The VDRL and liposome reagents discussed above are both composed of lipid-containing particles. Several types of rigid macromolecular particles have also been used in producing particle-agglutination reagents. For example, the Rapid Plasma Reagin (RPR) test, another non-treponemal syphilis test, employs a suspension of cardiolipin coated charcoal particles which are agglutinated in the presence of reagin antibody. The RPR teest is perfomed by mixing the charcoal suspension and the reagent sample, which may be an unheated serum or plasma sample, in a well-defined volume ratio on a plastic-coated card or the like. The card is rotated at about 100 rpm for 8 minutes to produce reagent-mediated charcoal particle agglutination. Agglutination can be determined visually, i.e. with the unaided eye.
The RPR test just described provides certain advantages over the VDRL syphilis test. The antigen-charcoal suspension is generally stable for a period of up to about a year, and thus fresh antigen suspensions need not be prepared. Sample preparation is easier, in that unheated serum or plasma samples may be used. The sample result can be read more easily and quickly since microscopic examination of the agglutination reaction is not required.
Nonetheless the RPR test has not been entirely satisfactory. The suspension and assay sample must be added together in a precisely defined volume ratio in order to maximize test sensitivity without producing false positives. This requirement increases the chance of false positive readings due to inadvertent volume-measuring errors, and requires that the technician periodically calibrate the needle used to deliver the antigen suspension. That the reaction must be rotated at a defined mixing speed for a relatively long mixing period (8 minutes) is a disadvantage as well.
Glass beads and polymeric microspheres, such as latex beads, are other types of rigid, macromolecular particles used in forming particle-agglutination reagents. Kakimi et al. have described another type of rigid-surface agglutination particle composed generally of a lipid core encased in a polymerized outer coat (reference 2). A disadvantage of these particles is that many ligands, including some lipophilic ligands, cannot be attached to glass or polymeric surfaces without significant loss in analyte-binding activity. Further, agglutination reactions involving rigid-surface agglutination particles may be relatively slow and therefore unsuitable for agglutination assays which are most conveniently performed on the surface of a glass slide or the like.
One general object of the invention, therefore, is to provide a novel large-liposome agglutination reagent which substantially overcomes above-discussed problems associated with particle-agglutination reagents known in the prior art.
A more specific object of the invention is to provide such a reagent which is adapted to produce particle agglutination, in the presence of a multivalent analyte within about 1 to 5 minutes.
Still another object of the invention is to provide such a reagent which contains a trapped dye at a concentration adapted to allow reagent agglutination to be visualized easily without magnification.
A particular object of the invention is to provide such a reagent for use in testing for syphilis.
Another particular object of the invention is to provide such a reagent for use in detecting an antigenic analyte such as hepatitis-B surface antigen.
Still another object of the invention is to provide an improved agglutination assay method which uses such a reagent.
The agglutination assay reagent of the invention includes liposomes predominantly in the 1-20 micron size range. The liposomes each contain a surface array of laterally mobile ligand molecules at a surface concentration adapted to produce reagent agglutination, within 1-5 minutes, when the reagent is incubated at room temperature with a multivalent ligand-binding analyte. In one embodiment of the invention, the reagent ligand includes cardiolipin at a lipid molar concentration of between about 1% and 20%.
The reagent also includes a dye trapped in the liposomes at a concentration adapted to allow reagent agglutination to be visualized easily without magnification. The dye may be anchored to the lipid components of the liposomes, or may be a water-soluble dye encapsulated in the liposome vesicular inner spaces. The quantity of reagent suspension which can be mixed with a given volume of analyte to produce optimal or near optimal agglutination without producing non-specific agglutination may be increased from a minimum optimal quantity, about four-fold.
An assay kit composed of a suspension of the lipid body reagent is stable for a period of up to about one year when stored under refrigeration.
These and other objects and features of the present invention will be more fully understood from the following detailed description.